Spinal apolipoprotein E is involved in inflammatory pain via regulating lipid metabolism and glial activation in the spinal dorsal horn

Introduction Inflammation and nerve injury promote astrocyte activation, which regulates the development and resolution of pain, in the spinal dorsal horn. APOE regulates lipid metabolism and is predominantly expressed in the astrocytes. However, the effect of astrocytic APOE and lipid metabolism on spinal cellular function is unclear. This study aimed to investigate the effect of spinal Apoe on spinal cellular functions using the complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model. Methods After intraplantar injection of CFA, we assessed pain behaviors in C57BL6 and Apoe knockout (Apoe−/−) mice using von Frey and Hargreaves’ tests and analyzed dorsal horn samples (L4-5) using western blotting, immunofluorescence, quantitative real-time polymerase chain reaction, and RNA sequencing. Results The Apoe levels were markedly upregulated at 2 h and on days 1 and 3 post-CFA treatment. Apoe was exclusively expressed in the astrocytes. Apoe−/− mice exhibited decreased pain on day 1, but not at 2 h, post-CFA treatment. Apoe−/− mice also showed decreased spinal neuron excitability and paw edema on day 1 post-CFA treatment. Global transcriptomic analysis of the dorsal horn on day 1 post-CFA treatment revealed that the differentially expressed mRNAs in Apoe−/− mice were associated with lipid metabolism and the immune system. Astrocyte activation was impaired in Apoe−/− mice on day 1 post-CFA treatment. The intrathecal injection of Apoe antisense oligonucleotide mitigated CFA-induced pain hypersensitivity. Conclusions Apoe deficiency altered lipid metabolism in astrocytes, exerting regulatory effects on immune response, astrocyte activation, and neuronal activity and consequently disrupting the maintenance of inflammatory pain after peripheral inflammation. Targeting APOE is a potential anti-nociception and anti-inflammatory strategy. Supplementary Information The online version contains supplementary material available at 10.1186/s13062-023-00444-z.


Introduction
Chronic pain resulting from injury and inflammation is a prevalent, persistent, and debilitating health concern [1].Spinal glial cells, such as astrocytes and microglia are reported to regulate nociception under chronic pain conditions [2].Astrocytes, which are a major glial cell type in the central nervous system (CNS), regulate neurotransmitter and calcium homeostasis, synapse formation, maturation, and elimination, blood-brain barrier function, and ion homeostasis, providing nutritional and trophic support to the brain [3,4].In particular, astrocytes exhibit enhanced lipid metabolism-related enzyme activity and are the major source of cholesterol and phospholipids in the CNS [5,6].Under pathological conditions, such as Alzheimer's disease (AD) and chronic pain, astrocytes are reactive and exhibit distinct metabolic features when compared with the resting astrocytes [7].Apolipoprotein E4 (APOE4), which is the strongest genetic risk factor for late-onset AD in humans, is reported to impair lipid metabolism in human astrocytes and microglia.Additionally, APOE4 upregulates matrisome pathway, chemotaxis, glial activation, and lipid metabolism in astrocytes through astrocyte-neuron communication [8].However, the role of astrocytic lipoprotein and lipid metabolism in the development of chronic pain is unclear.
APOE is a key regulator of lipid homeostasis and is primarily expressed in astrocytes in the brain [9].Additionally, APOE plays a crucial role in the metabolic network between astrocytes and neurons, transporting essential nutrients and antioxidants to the neurons [9][10][11].Furthermore, APOE is associated with neuroinflammation and synaptic plasticity in the nervous system [9,12,13].Among three APOE alleles/variants: APOE2, APOE3, and APOE4, in humans, the APOE4 allele is a genetic risk factor for developing AD, while APOE is also associated with chronic pain [14,15].The plasma levels of APOE are upregulated in patients with chronic low back pain [16].In contrast to humans, mice harbor a single Apoe allele [5].Animal studies have identified that Apoe in the spinal microglia and dorsal root ganglion (DRG) satellite glial cells mediates neuropathic pain [15,17].APOE is predominantly expressed and secreted by astrocytes in the spinal cord [12,17,18].Thus, this study aimed to investigate the potential role of astrocytic Apoe in the pathological processes of chronic pain and its regulatory effects on spinal cellular function under chronic pain conditions.
This study investigated the role of spinal astrocytic Apoe in in inflammatory pain in response to intraplantar complete Freund's Adjuvant (CFA) injection.We observed astrocytic Apoe levels in the spinal dorsal horn after CFA treatment.We further compared nociceptive responses and neuronal activity of spinal nociceptive neurons in control mice and Apoe −/− mice when subjected to CFA treatment.RNA sequencing and transcriptomic analysis, along with immunofluorescence revealed differential cellular changes in CFA-treated Apoe −/− mice.Finally, we observed the effect of the intrathecal injection of Apoe antisense oligonucleotide (ASO) on CFA-induced inflammatory pain hypersensitivity.These findings indicate the unique role of spinal astrocytic APOE in inflammation-induced pain and enhance our understanding of lipid metabolism in astrocytes in chronic pain conditions.

Animals
Male C57BL/6 J (C57) and Apoe −/− mice (B6/JGpt-Apoeem1Cd82/Gpt, Cas9-knockout, C57BL/6JGpt, T001458) aged 7-8 weeks with a bodyweight of 20-25 g were obtained from Gem-pharmatech Co., Ltd (Nanjing, China).All mice were housed in the animal facility of Xi`an Jiaotong University under the following conditions: temperature, 22-25 °C; circadian cycle, 12-h lightdark cycle.Animal experiments were approved by the Institutional Animal Ethics Committee of Xi'an Jiaotong University Health Science Center (No. 2018-334), ensuring compliance with legal and ethical guidelines.Strict guidelines have been adhered to minimize the use of mice and reduce animal suffering during the experiment.

Inflammatory pain model establishment
The CFA-induced inflammatory pain model was established by administering CFA into the left hind paw plantar of mice.An equal volume of CFA (Cat#: 344289; Sigma-Aldrich, St. Louis, MO, USA) and physiological saline were mixed to generate an oil-saline (1:1) emulsion for administration.Each mouse in the CFA group was intraplantarly administered with 10 μL of the prepared emulsion, while that in the control group was intraplantarly administered with 10 μL of physiological saline.
To establish the formalin-induced inflammatory pain model, a 5% formalin solution was prepared using a 10% neutral buffered formalin solution (Cat#: HT501128; Sigma-Aldrich) in physiological saline (1:1 ratio).The 5% formalin solution was subcutaneously injected into the left paw plantar.The control mice were injected with NS.The nociceptive behaviors, including paw withdrawal, licking, and paw flinching, were recorded at different time points after formalin administration.

Behavioral tests
The von Frey test was performed to quantify mechanical allodynia and mechanical hyperalgesia in mice using von Frey filaments weighing 0.16 g and 0.4 g, respectively [17,19].Mice were allowed to acclimatize to the environment for at least three days before behavioral experiments.Briefly, the unrestrained mice were transferred to a rectangular Plexiglass chamber placed on an elevated mesh platform after a 30-min adaptation period.Each filament was applied to the bottom of the hind paws 10 times.A positive response was recorded if the mouse exhibited sudden paw withdrawal, licking, or shaking.The percentage of paw withdrawal responses was calculated and expressed as paw withdrawal frequency (PWF, %).
Hargreaves' test was used to assess the thermal hypersensitivity of mice with an Analgesic Meter (Model 37370, Ugo Basile, Gemonio, Italy) [17].After acclimation for 30 min, the paw withdrawal latency (PWL) value was automatically recorded from the moment the heat source was activated until a strong paw withdrawal response was recorded.Each paw was tested five times at 5-min intervals.The average PWL was calculated for each paw in one trial.The cutoff time was set to 20 s to avoid tissue damage.

Paw edema evaluation
The paw volume was measured using an IITC 520 plethysmometer (IITC Life Science Instruments, CA).Measurements were recorded at baseline (V 0 ) and at different time points (V I ) post-CFA administration.To measure the paw volume (in mL), the paw was placed into a water bath during measurement.The paw volume (mL) was displayed on the readout and was used to determine the degree of paw edema.Paw edema was assessed by calculating the percentage of increase in paw volume after inflammation as follows: paw edema = [(V I − V 0 )/ V 0 ] × 100.

Intrathecal injection of ASO via lumbar puncture
The sequences of Apoe ASO described in a recent study and negative control (NC) ASO were GGT GAA TCT TTA TTA AAC and CCT ATA GGA CTA TCC AGG AA, respectively [17].ASOs were synthesized by GenePharma (Shanghai, China) and dissolved in sterile 0.01 M phosphate-buffered saline (PBS; PH 7.4) immediately before administration.To inhibit RNase H-mediated degradation and promote cellular uptake, the ASOs were subjected to 2′-O-methoxyethyl modifications on the 5′-terminus and 3′-terminus and phosphorothioate modifications.The mice were intrathecally administered with ASO or PBS 3 days before CFA administration.The injection was performed at the L4-L5 posterior intervertebral space using a sterile insulin needle syringe under inhalational anesthesia via 3% isoflurane.To enhance the spread of the injected solution around the spinal cord and prevent efflux to the periphery, the needle was not moved for 30 s after the injection.

Western blotting
The spinal dorsal horn from the L4-L5 spinal segment was collected after the mice were decapitated under isoflurane anesthetization.The tissues were homogenized in cold radioimmunoprecipitation assay lysis buffer (Cat#: P0013D; Beyotime, Shanghai, China) with phenylmethanesulfonyl fluoride (100 mM; Cat#: ST506; Beyotime) and protease inhibitor cocktail (100 × , Cat#: HY-K0010; MedChemExpress, Shanghai, China).The lysates were centrifuged at 1000 g and 4 °C for 15 min.The supernatant was used as the cytosolic fraction and subjected to western blotting.The protein concentration was determined using the bicinchoninic acid assay (Cat#: P0010; Beyotime).The samples were incubated at 99 °C for 5 min and loaded onto a 10% stacking/7.5% separating sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel quick preparation kit (Cat#: P0012AC; Beyotime).The resolved proteins were transferred to a nitrocellulose membrane (pore size: 0.45 μm).The membrane was blocked with QuickBlock ™ blocking buffer (Beyotime) and probed with rabbit polyclonal anti-Apoe antibodies (purified full-length native protein corresponding to mouse Apoe; 1:1000; Cat# ab20874; Abcam, Cambridge, MA, USA) or mouse anti-β-actin antibodies (1:2000; Cat#: sc-8432; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight.After washing thrice with Tris-buffered saline with 0.05% Tween 20 (TBST), the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG (1:20,000; Cat#: A9169; EMD Millipore, Darmstadt, Germany) or goat anti-mouse IgG (H + L) (1:20,000; Cat#: DY60203; DEEYEE, Shanghai, China).Immunoreactive signals were developed using an enhanced chemiluminescence (ECL) kit (Clarity Western ECL Substrate; Cat#: WBKLS0500; EMD Millipore) and digitized into a digital film using a Champchemi System with SageCapture software (Sagecreation Service for Life Science, Beijing, China).The densitometric images were analyzed using ImageJ Software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).The β-actin was used as an internal control.The average value in the saline-treated groups was set to 100% after the expression of Apoe was normalized to that of β-actin.The relative Apoe level was presented as a ratio of the normalized value to the average value of all saline-treated groups.

Immunofluorescence analysis
Mice were anesthetized and transcardially perfused with physiological saline, followed by perfusion with 4% paraformaldehyde (PFA) in 0.01 M PBS.The whole spinal cord was excised, post-fixed overnight in the same solution, and dehydrated using gradient sucrose solution (20% and 30%) until it sank at 4 °C.The L4-L5 spinal cord was dissected and sliced into sections with a thickness of 30 μm using a Leica CM1860 cryostat (Leica, Wetzlar, Germany).For single labeling, the sections were washed with 0.01 M PBS, preincubated with blocking buffer (0.01 M PBS supplemented with 0.3% Triton X-100 and 10% goat serum) for 1 h at 25 °C, and incubated with rabbit polyclonal anti-Apoe (1:500; Cat#: ab20874; Abcam), rabbit polyclonal anti-Fos (1:10,000; Cat#: ab190289; Abcam), rabbit IgG anti-p-Jun (Ser63; p-c-Jun) (1:200; Cat#: CY6576; Abways, Shanghai, China), or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibodies (clone G-A-5, purified from hybridoma cell culture; 1:500; Cat#: G6171; Sigma-Aldrich) overnight at 4 °C.After washing thrice with PBS, the sections were incubated with donkey Alexa Fluor 488-conjugated antirabbit secondary antibodies (1:200; Cat#: ab150073; Abcam) for Apoe and Fos, Cy3-conjugated goat antirabbit secondary antibodies (1:500; Cat#: AP132C; EMD Millipore) for p-Jun, or Cy3-conjugated goat anti-mouse secondary antibodies (1:500; Cat#: AP124C; EMD Millipore) for GFAP at 4 °C for 2 h.For double labeling of Apoe and GFAP, the sections were incubated with the rabbit anti-Apoe antibody and mouse anti-GFAP antibodies, followed by incubation with a mixture of the corresponding secondary antibodies.The microscopic images were captured using an Axio Scope A1 fluorescence microscope (ZEISS, Carl Zeiss, Germany) and processed and quantified using ImageJ Software (Wayne Rasband).The Apoe-positive, GFAP-positive, or p-Junpositive areas and the total area of the spinal dorsal horn were quantified and the percentage of positive area in the partial or whole spinal dorsal horn was calculated.The correlation between the colocalization of Apoe and GFAP was examined using Pearson's correlation coefficient as described previously [17].Briefly, the images of the double-labeled sections were split according to the channels using Fiji's "Coloc 2" plugin in Image J Software.The fluorescence signals were measured after the segmentation of the background by thresholding.Pearson's r-value will be shown in a PDF file after the channel 1 and 2 images were selected as red and green signals, respectively.A strong correlation between the two labeled signals was indicated when the r-value was greater than 0.5.For immunostaining, all images from at least 3 mice were randomly chosen.The minimal number of images from one mouse was 3.

RNA sequencing and bioinformatics analysis
At 24 h post-bilateral intraplantar injection of CFA, bilateral L4-L5 spinal dorsal horns were dissected from C57 or Apoe −/− mice subjected to rapid decapitation under isoflurane anesthetization.The L4-L5 spinal dorsal horn from one mouse was treated as an independent biological replicate.In total, 8 samples (four independent biological replicates per group) were sent to BGI Genomics Inc. (Wuhan, China).RNA sequencing was performed as described previously [20].Briefly, total RNA was extracted and purified according to the manufacturer's instructions (BGI Genomics).RNA sequencing was outperformed using an Illumina Hiseq × 10 platform High Output Mode in a 50-bp paired-end configuration with at least 20 million (M) reads per sample (BGI Genomics).Eight samples were then subjected to multiplexing, sequencing, differential gene expression analysis, and transcript expression analysis.The gene expression levels between the groups were compared using Student's t-test and transformed into log 2 values.Differentially expressed genes (DEGs) were then extracted based on the criterion adjusted P < 0.05 and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using DAVID Bioinformatics Resources 6.7 with the phyper function in R software.Enrichment was considered significant at Q ≤ 0.05.

Statistical analysis
All statistical analyses were performed using GraphPad Prism 9 (GraphPad, San Diego, CA, USA).Experimental animals were randomized and assigned to various treatment groups.For behavioral tests, 6-10 mice were included in each group.Meanwhile, four mice were included in each group for RNA sequencing analysis of bilateral spinal cord dorsal horn.Three independent qRT-PCR and western blotting experiments were performed for statistical analysis of different biological repeats.The correlation of double-label immunofluorescence analysis results was examined using Pearson's correlation coefficient.

CFA upregulated Apoe expression in the lumbar spinal dorsal horn
The results of the Hargreaves test illustrated that CFA injection into the plantar surface of the mice' left hind limb provoked thermal hyperalgesia, characterized by an obvious decrease in the PWL of the ipsilateral hind paw tested at 2 h and on day 1 and 3 after the injection, compared to the baseline control before CFA administration or saline-treated mice (Fig. 1A).Expectedly, the intraplantar saline injection resulted in no significant differences in PWL at the tested time points compared with the baseline control before saline injection (Fig. 1A).The results of western blotting demonstrated that Apoe expression was markedly upregulated (approximately 1.67-fold of ) at 2 h post-CFA injection compared to the saline-treated mice (two-tailed unpaired Student's t-test) (Fig. 1B).The expression of Apoe protein continued to increase on days 1 (approximately 2.36-fold) and 3 (approximately 2.64-fold) post-CFA treatment but partially recovered on day 7 (approximately 1.66-fold) post-CFA treatment (Fig. 1B).Additionally, the Apoe-positive area in the contralateral spinal dorsal horn increased at all tested time points post-CFA treatment (Fig. 1C).The upregulated Apoe expression was further confirmed using immunofluorescence.Immunofluorescence analysis revealed that the Apoe-positive area in the ipsilateral spinal dorsal horn of CFA-treated mice was higher than that in the ipsilateral spinal dorsal horn of saline-treated mice on day 1 post-CFA treatment (Fig. 1D-F).Pearson's correlation coefficient value for the double labeling of Apoe and GFAP was similar between saline-treated and CFA-treated mice (Fig. 1G, H), suggesting that astrocytes were the primary source of Apoe production at the early stage (day 1) of CFA-induced inflammatory pain.CFA did not affect the spinal dorsal horn mRNA levels of Apoe (Fig. 1I).However, the spinal Apoe protein level was upregulated in the early stages of CFA-induced inflammatory pain.This suggests that Apoe is involved in the development of inflammatory pain.

The less pain hypersensitivity in Apoe −/− mice in response to CFA injection
To determine the role of spinal Apoe in the development of inflammatory pain, CFA-induced pain hypersensitivity in Apoe −/− and C57 mice was comparatively analyzed.The complete knockout of Apoe in mice was verified using immunofluorescence staining.Apoe −/− mice did not exhibit Apoe immunostaining signals in the spinal cord (Fig. 2A).In C57 mice, the PWF in response to stimulation with 0.16 and 0.4 g von Frey filaments were higher (Fig. 2B, C) and PWLs to thermal stimulation were lower (Fig. 2D) on the ipsilateral sides in the CFAtreated group than those in the saline-treated group at 2 h and on day 1 post-CFA treatment.These data indicate that CFA treatment sufficiently induced mechanical and thermal pain hypersensitivity in C57 mice.However, Apoe −/− mice exhibited decreased PWF (Fig. 2B, C) and prolonged PWL (Fig. 2E) when compared with C57 mice on day 1, but not at 2 h, post-CFA treatment.This indicates that Apoe −/− mice did not exhibit persistent pain hypersensitivity.Thus, Apoe may be involved in the transition from acute pain to persistent pain after peripheral inflammation.Additionally, PWFs or PWLs on the contralateral sides were similar between the groups (Fig. 2E-G), indicating that Apoe deficiency did not affect basal nociceptive responses to either mechanical or thermal stimulation.Next, paw edema in CFA-treated C57 and Apoe −/− mice was comparatively analyzed.Paw edema was evaluated by measuring the percentage increase in paw volume using an IITC 520 plethysmometer.In this study, paw edema was not observed in saline-treated mice (Fig. 2H).CFA treatment induced paw edema in C57 mice at 2 and 24 h post-treatment (Fig. 2H).Paw volume was similar between Apoe −/− and C57 mice at 2 h post-CFA treatment.Similar to nociceptive responses, paw edema in Apoe −/− mice was significantly alleviated when compared with that in C57 mice at 24 h post-CFA (Fig. 2H).
To further verify the effect of spinal Apoe knockout on acute inflammatory pain, formalin-induced acute pain in Apoe −/− mice was examined.Paw withdrawal time (Fg.2I), licking time (Fig. 2J), or flinching and shaking numbers (Fig. 2K) were not significantly different between Apoe −/− and C57 mice.

The less Fos expression in Apoe −/− mice in response to CFA injection
Next, this study investigated the effect of spinal Apoe on neuronal hyperexcitability by evaluating the Fos expression levels in the spinal dorsal horn of CFA-treated Apoe −/− mice.On day 1 post-CFA treatment, the mouse paw was brushed repeatedly for 1 min to induce Fos expression in the spinal dorsal horn.In C57 mice, the number of Fos-positive neurons in both superficial (Layers I-III) and deep dorsal horns (Layers IV-VI) in the CFA-treated group was significantly higher than that in the saline-treated group (Fig. 3A-C).The number of Fos-positive neurons in CFA-treated Apoe −/− mice was significantly lower than that in CFAtreated C57 mice (Fig. 3A-C).This indicates that spinal Apoe deficiency impaired noxious stimulus-induced spinal neuron excitability.To investigate the potential molecular mechanism through which Apoe mediates spinal neuron hyperexcitability and inflammatory pain, RNA sequencing was performed to identify differentially expressed mRNAs between CFA-treated C57 and Apoe −/− mice.In total, 63 DEGs were identified between CFA-treated C57 and Apoe −/− mice (Q < 0.05) (Fig. 4A, Additional file 1: Excel 1).Furthermore, hierarchical clustering analysis revealed 26 DEGs (log 2 -(fold change) > 0.5 or < − 0.5) (Fig. 4B).GO enrichment analysis revealed that the 63 DEGs were enriched in different level 2 GO terms as follows: biological process term: synapse pruning, antigen processing and presentation, immune response, cholesterol metabolic process, microglial cell activation, and positive regulation of T cell activation (Fig. 4C; Additional file 2: Excel 2); molecular function term: binding, transporter activity, transcription regulator activity, and catalytic activity (Fig. 4D; Additional file 3: Excel 3).In the level 2 GO term binding function, the DEGs could bind with major histocompatibility (MHC) class II protein complex, growth factors, peptide antigens, high-density lipoprotein particles, CD4 receptors, polysaccharides, signaling receptors, low-density lipoprotein particles, and insulin-like growth factor (Fig. 4D).Meanwhile, in the level 2 term transporter activity, the DEGs were involved in transmembrane cholesterol transfer activity, transmembrane transport of L-amino acids, and lipid transport activity (Fig. 4D).
Protein-protein interaction (PPI) network of 63 DEGs revealed that Apoe was associated with Jun, Cx3cr1, and C1qa (Fig. 4F).Cx3cr1 is a receptor that is specifically expressed in microglia [21].MAPK8, which is specifically expressed in spinal astrocytes and is required for the maintenance of bilateral mechanical allodynia induced by CFA, activates Jun [22].These findings indicate that Apoe deficiency exerts antinociceptive effects by significantly modulating glial function under inflammatory pain conditions.

The less astrocyte activation in Apoe −/− mice in response to CFA injection
Next, the regulatory effect of Apoe on astrocyte activation in the spinal dorsal horn of the CFA-induced inflammatory pain mouse model was examined.The p-Jun-positive and GFAP-positive areas were upregulated in the ipsilateral spinal dorsal horn of CFA-treated C57 mice (Figs. 5 and 6).However, the CFA-induced upregulation of p-Jun-positive and GFAP-positive areas in the ipsilateral and contralateral spinal dorsal horns was mitigated in Apoe −/− mice (Figs. 5 and 6).These findings suggest that the antinociceptive effect of Apoe deficiency can be attributed to its inhibitory effects on astrocyte activation under CFA-induced inflammatory pain conditions.

Intrathecal injection of Apoe ASO alleviates CFA-induced inflammatory pain
Previously, Apoe ASO was reported to downregulate Apoe expression in the DRG [17].The microinjection of Apoe ASO into the DRG or the intrathecal injection of Apoe ASO exerted antinociceptive effects on spinal nerve ligation-induced neuropathic pain [17].This study examined the effect of Apoe ASO on CFA-induced inflammatory pain.ASO was intrathecally administered three days before CFA injection and the effect of ASO on pain hypersensitivity in CFA-treated mice was examined.CFA induced apparent mechanical allodynia and heat hyperalgesia on the ipsilateral paws at all tested time points after CFA injection (Fig. 7A-C), except for day 3 post-CFA treatment (partial recovery of heat hyperalgesia was observed) (Fig. 7C).The intrathecal injection of Apoe ASO dose-dependently attenuated CFA-induced mechanical allodynia (Fig. 7A, B) and heat hyperalgesia on days 1 and 3, but not at 2 h, post-CFA injection (Fig. 7C) on the ipsilateral side of ASO-treated mice.
Treatment with 66 µg ASO produced the maximal effect on CFA-induced pain hypersensitivity (Fig. 7A-C).The intrathecal injection of ASO or vehicle did not affect the basal paw withdrawal responses on the contralateral side relative to the baseline level (Fig. 7D-F).Finally, the spinal tissues were harvested after behavioral tests and subjected to western blotting to analyze Apoe expression.Apoe ASO downregulated Apoe expression by 40% (Fig. 7G).These findings suggest that the intrathecal treatment of Apoe ASO exerts therapeutic effects on chronic inflammatory pain.

Discussion
In this study, Apoe was upregulated in the spinal astrocytes of the CFA-induced inflammatory pain mouse model.Compared to the C57 mice, Apoe −/− mice exhibited decreased pain hypersensitivity and paw edema inresponse to CFA injection.Consistently, the spinal dorsal horn Fos immunoreactivity, which serves as an indicator of neuronal activation [23,24], in Apoe −/− mice was less responsive when compared with that in C57 mice after CFA treatment.RNA sequencing and immunostaining analysis revealed spinal cellular changes associated to lipid metabolism and the glial activation in CFA-treated Apoe −/− mice which may underlie the anti-nociceptive effect of Apoe deficiency.Additionally, treatment with Apoe ASO, which downregulates Apoe expression, decreased pain hypersensitivity in CFA-treated mice.Pain alleviation was not observed in Apoe −/− or Apoe ASO-treated mice at the early stage (2 h) of inflammation.This study demonstrated the crucial role of Apoe in persistent inflammatory pain.Apoe along with high-density lipoproteins mediates lipid transport and cholesterol homeostasis in the CNS [25].APOE is primarily expressed in astrocytes in both the brain and the spinal cord, as well as in the glia surrounding the sensory neurons in DRGs, and the nonmyelinating Schwann cells of peripheral nerves [9,17,26].The activation of microglia and astrocytes in the spinal cord is considered a key mechanism underlying the genesis of chronic pain [27].This could explain the role of spinal astrocytic Apoe in the development of chronic pain.This study demonstrates that Apoe deficiency has   antinociceptive effects during the late stages of inflammatory pain.Our previous study has demonstrated that Apoe in DRG glial cells is involved in the development of neuropathic pain in mice [17].Additionally, as the essential role of Schwann cells in the development of neuropathic pain [28], Apoe in non-myelinated Schwann cells may mediate neuropathic pain.It requires further investigation.We also note that Apoe knockout has not been shown to have an effect on the basal nociceptive response, or on the acute phase of inflammatory pain.Moreover, we previously observed that the peripheral or intrathecal application of Apoe did not result in any nociceptive responses (licking, paw flicking, and paw withdrawing) [17].This may be explained that acute pain was produced majorly associated with acute inflammation induced by inflammatory mediators (such as bradykinin, prostaglandins, H + , ATP, and nerve growth factor), proinflammatory cytokines, and chemokines released locally from neutrophils, macrophage, T cells, and mast cells [27].
RNA sequencing data revealed that Apoe knockout in the spinal dorsal horn exerts regulatory effects on synapse pruning, cholesterol metabolic processes, immune response, and activation of glial cells and T cells.Immunofluorescence analysis demonstrated that Apoe knockout suppresses astrocyte activation under CFA-induced inflammatory pain conditions.These data indicate the mechanism through which spinal astrocytic Apoe modulates pain processing under persistent inflammatory pain conditions.Astrocytes are the major source of lipids, providing nutritional support for the brain neurons [5,6].The lipid droplets in astrocytes play an essential physiological and protective role in the CNS [6].Astrocytes release APOE that transports lipids and couples metabolism between astrocytes and neurons [6,10].Under pathological conditions, such as AD, acute stroke, and epilepsy, astrocytes can take up lipids from hyperactive neurons to exert protective effects against lipid toxicity, resulting in excessive astrocytic lipid accumulation and lipid metabolism reprogramming [29,30].The newly discovered subtype of reactive astrocytes, which are rich in lipids and exhibit the neurotoxic phenotype, induces cell death via saturated lipids contained in APOE lipoparticles to mediate astrocyte-induced toxicity [30,31].Astrocytes can take up toxic fatty acids secreted from hyperactive neurons in the form of APOE lipid-containing particles, which are stored in lipid droplets and metabolized in the mitochondria through β-oxidation [10,29].Thus, we hypothesize that Apoe upregulation in astrocytes of the spinal cord after CFA Previous studies have reported that Apoe is mainly expressed in astrocytes it also has very low expression in microglia, as demonstrated in several studies [15,17,26].In our RNA sequencing data, we noticed an increase in cx3cr1 transcript levels in Apoe(-/-) mice compared with C57 mice.The cx3cr1 gene encodes CX3CR1, which is primarily expressed in microglia and macrophages and has been implicated in neuropathic pain by affecting microglia-neuron crosstalk in the spinal dorsal horn [21,32,33].Apoe is the top upregulated gene in spinal cord microglia at chronic time points in a peripheral nerve injury-induced neuropathic pain mouse model [15].Additionally, Apoe was a hub gene in both macrophages and microglia in the subacute and chronic phases of spinal cord injury [34].In mice, astrocytes with Apoe knockout exhibited upregulated matrisome pathway, chemotaxis, and inflammation.Meanwhile, Apoe knockout microglia exhibited upregulated cholesterol biosynthesis and other lipid pathways, predicting upregulated lipid synthesis [8].Cholesterol metabolism in spinal microglia is associated with chemotherapy-induced neuropathic pain [35].The long-lasting therapeutic effect of apolipoprotein A-I binding protein on neuropathic pain via cholesterol depletion was associated with antiinflammatory effects and cholesterol metabolism reprogramming and decreased accumulation of lipid droplets in microglia [35].Thus, APOE modulates the function of microglia in the spinal cord and is involved in CFAinduced inflammatory pain processing.
APOE mediates the transfer of cholesterol from astrocytes to neurons to form membranes, synaptic vesicles, and clusters of postsynaptic receptors [12,25,36].Previous studies have demonstrated that the brain of APOE4 allele carrier mice exhibits decreased dendritic length, spinal density, variable synaptic transmission, and longterm potentiation patterns, as well as the downregulation of synaptic proteins, such as Syp and Stx1a [36][37][38].Furthermore, APOE4 mice exhibited increased synaptic protein loss after lipopolysaccharide injection as evidenced by the downregulation of PSD-95, drebin, and synaptophysin [39].Ibuprofen, a well-known nonsteroidal anti-inflammatory drug, exerts antinociceptive effects and can counteract the inhibitory effect of the APOE4 genotype on dendritic spine density [37].In this study, the less neuronal activity of spinal nociceptive neurons occurred in Apoe −/− mice after CFA injection.Several DEGs associated with synapse pruning were identified in CFA-treated Apoe −/− mice.Thus, we hypothesized that Apoe knockout impairs the transport of cholesterol and lipids to the neurons.Consequently, the generation of new membranes in neurons, which is necessary for axon extension and dendritic spine production, is downregulated, contributing to pain alleviation in Apoe −/− mice.
Humans have three APOE alleles/variants (APOE2, APOE3, and APOE4) [40].The APOE4 allele is the most important genetic risk factor for developing AD in humans [14,41].Among men, APOE4 allele carriers have a decreased risk of developing chronic pain, whereas APOE2 allele carriers are at an increased risk of developing chronic pain [15].However, the APOE2 allele is not correlated with chronic pain in women [15].Furthermore, the APOE4 allele is a protective factor against migraines and stress, whereas the APOE2 allele is a risk factor [42]. Human APOE isoforms differ at amino acid positions 112 and 158 (cysteine at positions 112 and 158 in APOE2, a cysteine at position 112 and an arginine at 158 in APOE3, and arginine at positions 112 and 158 in APOE4) [43].APOE4 is the risk factor for AD due to its toxic effect on lipid metabolism in microglia and astrocytes [8,10].Further studies are needed to understand the distinct roles of human APOE variants in different pathological conditions.In contrast to humans, mice harbor only a single Apoe allele [5].The "target replacement" models of human APOE variants have been developed by replacing the Apoe allele of mice with one of the human APOE isoforms [44,45].These APOE target replacement mouse models can be used to study the differential role of human APOE isoforms in chronic pain.

Conclusions
This study demonstrated the critical role of spinal Apoe in the maintenance of inflammatory pain.Apoe knockout exerts regulatory effects on the function of astrocytes, microglial cells, and neurons, alleviating inflammatory pain (Fig. 8).Apoe knockout may downregulate cholesterol synthesis in astrocytes, preventing the accumulation of cholesterol and lipids in astrocytes and consequently suppressing astrocyte activation.Thus, APOE can be a potential target for developing anti-nociception and antiinflammation strategies.• thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 1
Fig. 1 Complete Freund's adjuvant (CFA) upregulates Apoe protein expression in the spinal dorsal horn.A Paw withdrawal latency (PWL) to thermal stimulation in mice after physiological saline or CFA treatment.N = 6-16 mice/group.**p < 0.01, ****p < 0.0001 versus the saline-treated mice by two-way analysis of variance (ANOVA) followed by post hoc Tukey test.B-C Representative blots and quantification of Apoe expression in the ipsilateral (A) and contralateral spinal dorsal horns (B) after saline or CFA treatment.N = 4-5 mice/group.## p = 0.001 compared with saline-treated mice (two-tailed unpaired Student's t-test).* p = 0.0117, ** p = 0.0020 compared with saline-treated mice (one-way ANOVA, followed by Tukey's post-hoc test).D-F Immunofluorescence staining (C) and quantification (D, E) of Apoe in the ipsilateral (Ipsi; E) and contralateral (Contra; F) spinal dorsal horns on day 1 post-saline or CFA treatment.N = 15 sections from 3 mice per group.The individual dot indicates one value from one section.All dots in one cluster present all values from one mouse.** p = 0.003 compared with saline-treated mice (two-tailed unpaired Student's t-test).Bar: 100 μm.G-H Colocalization of Apoe with GFAP and Pearson correlation coefficients (r) between Apoe staining and GFAP staining.N = 26 sections from 3 mice per group.Bar: 50 μm.I The mRNA level of Apoe in the spinal dorsal horn on day 1 post-saline or CFA treatment.N = 4 mice/group.Two-tailed unpaired Student's t-test

FosFig. 3
Fig. 3 Complete Freund's adjuvant (CFA) downregulates Fos expression in the spinal dorsal horn of Apoe −/− mice.The left hind paw of all mice was repeatedly brushed for 1 min two hours before cardiac perfusion.A Immunofluorescence analysis of Fos expression in the three groups.B-C Quantification of Fos-positive neurons in the superficial dorsal horn (Layers I-III, b) and deep dorsal horn (Layers IV-VI, c).N = 3 mice/group (3-5 sections/mouse).**** p < 0.0001 compared with physiological saline-treated C57 mice; ## p = 0.0012, ### p = 0.0002 compared with CFA-treated C57 mice.Two-way analysis of variance, followed by Tukey's post-hoc test

Fig. 4
Fig. 4 Differentially expressed genes (DEGs) between Apoe −/− and C57BL6 (C57) mice after complete Freund's adjuvant (CFA) treatment.A Volcano plot of 63 DEGs between CFA-treated C57 and Apoe −/− mice.B Heatmap of 26 DEGs determined based on log 2 (fold change (FC)) > 0.5 or < − 0.5.C The enrichment of DEGs in the Gene Ontology (GO) term molecular function.D The enrichment of DEGs in the GO term biological process.E Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 63 DEGs.F Protein-protein interaction (PPI) network of 63 DEGs, including Apoe, Jun, Cx3cr1, and C1qa

Fig. 7
Fig. 7 Apoe knockdown via intrathecal administration of Apoe antisense oligonucleotide (ASO) suppresses complete Freund's adjuvant (CFA)-induced pain hypersensitivity.Apoe ASO was intrathecally administered 3 days and 1 h before CFA treatment.The behavioral tests were performed before Apoe ASO treatment and at 2 h and on days 1 and 3 post-CFA treatment.A-F The effect of Apoe ASO on CFA-induced pain hypersensitivity to 0.16-g von Frey filament (A, D), 0.4-g von Frey filament (B, E), and thermal stimuli (C, F) on the ipsilateral (A-C) and contralateral sides (D-F) after CFA treatment.N = 5-11 mice/group.**** p < 0.0001 and *** p = 0.0006 compared with phosphate-buffered saline (PBS) and CFA-treated mice at 2 h post-treatment; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with CFA and PBS-treated mice.Two-way analysis of variance, followed by Tukey's post-hoc test.G The effect of Apoe ASO on Apoe expression in the spinal dorsal horn.N = 6 mice/group.** p = 0.0013 compared with CFA and PBS-treated mice (two-tailed unpaired Student's t-test)

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